Effect of silymarin on the relative gene expressions of some inflammatory cytokines in the liver of CCl4-intoxicated male rats

Abstract

The intensive exposure of the liver cells to any type of noxae, such as viruses, drugs, alcohols, and xenobiotics could induce hepatic inflammation through the upregulation of gene expression of several fibrotic and inflammatory mediators. So, our study assessed the role of silymarin on the inflammatory response induced by carbon tetrachloride (CCl₄) as an example of xenobiotics on liver tissues in male rats. Forty-eight Wister male rats (weight: 130 ± 10) were housed for 14 days and then divided randomly into six groups: control, SLY: rats received only silymarin orally for 12 weeks (daily), CO: rats were injected with corn oil for 8 weeks (3 times weekly), CCl₄: rats were injected with CCl₄ solubilized in corn oil for 8 weeks (day by day), Treated: rats received silymarin for 4 weeks after CCl₄ injection, Protected: rats received silymarin for 4 weeks before and 8 weeks during CCl₄ injection. When the treatment period for the rats was over, they underwent scarification after anesthesia. Then, the sera were extracted from the collected blood for the determination of irisin levels, liver functions, and lipid profiles. Liver tissues were separated for the histopathological examinations, the determination of oxidative stress (OS) parameters content, and the relative gene expression of inflammatory cytokines; nuclear factor kappa (NF)-κB, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, cyclooxygenase (COX)-2, and transforming growth factor beta (TGF-β). The findings showed that silymarin reduced liver inflammation by overcoming the OS process and inflammatory cytokines production which was stimulated by CCl₄. These results were confirmed by histopathology of liver tissues.

Figure 1

Experimental design flowchart.

Figure 2

Microscopic examination of liver tissues stained by H&E; (A) Ctrl group: control rat liver demonstrating the normal architecture of the central compartment of the hepatic lobule. The central vein (CV) occupies the core of the lobule. Strings of hepatocytes arrayed from the CV. They are departed by narrow sinusoids (s). The hepatocytes demonstrate a normal granular and eosinophilic cytoplasm. Hepatocytes have a single nucleus (h); (B) SLY group: Liver section from a rat receiving silymarin for 12 weeks revealing the unaltered organization of cords of hepatocytes radiating around the CV and focal aggregates of cellular infiltrates (arrow) are depicted around the portal tract (PT); (C) CO group: The PT of the Oil-treated rat liver demonstrating vacuolation of the hepatocyte cytoplasm (H). the portal vein (PV) tributary, the branch of the portal artery (PA), (b) = bile ductules, scanty stromal cells (arrow); (D) CCl₄ group: The PT of the liver of rat injected with CCl₄ induced lesion revealing consistent increased connective tissue stroma (black star) with dilation of biliary cholangioles (ch). Some hepatocytes have a single nucleus (h1); others are binucleated (h2). Pivotal clumps of cellular infiltrate (arrow) are depicted. (E) Treat-CCl₄ group: Treatment with silymarin after CCl₄-induced lesion depicting focal areas (arrows) with vacuolated hepatocytes (H1) among the majority of normal hepatocytes (H2). (F) Prot-CCl₄ group: Liver section of rat treated with silymarin before and during CCl₄ injection; showing recovery of hepatocytes (H3) and general architecture of the hepatic lobule. All images are original with microscopic magnification × 400.

Figure 3

Microscopic examination of liver tissue stained by Gomori’s trichrome stain; (A) Ctrl group: Negative control rat liver demonstrates the supporting stroma and minimal collagen fiber content (arrow) depicted in the PT. (B) SLY group: Liver section from a rat receiving silymarin for 12 weeks showing average connective tissue content (arrow pointing to greenish collagen fibers) (C) CO group: liver of rat treated with oil vehicle showing average connective tissue deposit (arrow) in the PT (D) CCl₄ group: Liver section from rat treated with CCl₄ induction of lesion illustrating non-resolving of the fibrous tissue (arrow pointing to green collagen fibers) deposited in the PT and the CV. Note the extensive diffusion of degenerated cells (yellow arrow) appeared between the hepatocytes. Abnormal inspissation of erthrocytes (*) noticed in the hepatic sinusoids as in the PT tributaries. (E) Treat-CCl₄ group: liver of rat treated with silymarin 4 weeks after CCl₄ induced lesion demonstrating persisting collagen bands arrow pointing to a greenish band of fibers) around the dilated central vein. (F) Prot-CCl₄ group: Liver section from rats treated with silymarin before and during CCl₄ induced lesion demonstrating an evident reduction in the greenish deposits of collagen fibers around the CV and the PT stroma. All images are original with microscopic magnification × 100.

Figure 4

Effect of silymarin administration on (A) the concentrations of Serum Irisin and the gene expression of the hepatic; (B) NF-κB, (C) TNF-α, (D) IL-6, (E) COX-2, and (F) TGF-β against CCl₄-injection. The findings are expressed as mean ± SD (n = 8). Different characters for the same parameter are significantly different at p ˂ 0.05.

Figure 5

Graphical abstract.